23-Hydroxybetulinic acid attenuates 5-fluorouracil resistance of colorectal cancer by modulating M2 macrophage polarization via STAT6 signaling

Abstract

Macrophage polarization is closely associated with the inflammatory processes involved in the development and chemoresistance of colorectal cancer (CRC). M2 macrophages, the predominant subtype of tumor-associated macrophages (TAMs) in a wide variety of malignancies, have been demonstrated to promote the resistance of CRC to multiple chemotherapeutic drugs, such as 5-fluorouracil (5-FU). In our study, we investigated the potential of 23-Hydroxybetulinic Acid (23-HBA), a significant active component of Pulsatilla chinensis (P. chinensis), to inhibit the polarization of M2 macrophages induced by IL-4. Our results showed that 23-HBA reduced the expression of M2 specific marker CD206, while downregulating the mRNA levels of M2 related genes (CD206, Arg1, IL-10, and CCL2). Additionally, 23-HBA effectively attenuated the inhibitory effects of the conditioned medium from M2 macrophages on apoptosis in colorectal cancer SW480 cells. Mechanistically, 23-HBA prevented the phosphorylation and nuclear translocation of the STAT6 protein, resulting in the inhibition of IL-10 release in M2 macrophages. Moreover, it interfered with the activation of the IL-10/STAT3/Bcl-2 signaling pathway in SW480 cells, ultimately reducing M2 macrophage-induced resistance to 5-FU. Importantly, depleting STAT6 expression in macrophages abolished the suppressive effect of 23-HBA on M2 macrophage polarization, while also eliminating its ability to decrease M2 macrophage-induced 5-FU resistance in cancer cells. Furthermore, 23-HBA significantly diminished the proportion of M2 macrophages in the tumor tissues of colorectal cancer mice, simultaneously enhancing the anti-cancer efficacy of 5-FU. The findings presented in this study highlight the capacity of 23-HBA to inhibit M2 macrophage polarization, a process that contributes to reduced 5-FU resistance in colorectal cancer.

Keywords: 23-Hydroxybetulinic acid; 5-fluorouracil; Chemoresistance; Colorectal cancer; Macrophage polarization; STAT6.

Fig. 1

23-HBA inhibited M2 macrophage polarization induced by IL-4. A Schematic diagram of experiment. B Analysis of the administration of 23-HBA (2.5, 5, 10, 20, 40 μM) on cell viability of M0 macrophages for 48 h. C, D M0 macrophages were treated with IL-4 (20 ng/mL) with or without 23-HBA (10, 20 μM) for 48 h. These treated cells were collected for the following analyses. C The expression of CD206 was analyzed by flow cytometry. D RT-PCR was performed to analyze the mRNA levels of CD206, Arg-1, IL-10, and CCL2. The GAPDH gene was used as internal control. Data were presented as mean ± SD (n = 3). Statistical significance: *p < 0.05, **p < 0.01

Fig. 2

23-HBA inhibited IL-4-induced phosphorylation and nuclear translocation of STAT6 in macrophages. M0 macrophages were treated with IL-4 (20 ng/mL) with or without 23-HBA (10, 20 μM) for 48 h. These treated cells were collected for the following analyses. A Relative protein levels of p-STAT6/STAT6, p-STAT3/STAT3, and p-JAK2/JAK2 were determined by western blot. β-tubulin was used as internal control. B Docking simulation results of 23-HBA and STAT6. C The nuclear translocation of STAT6 was detected by immunofluorescence. Bars represent 50 μm. D The expression of M2 marker CD206 was detected by immunofluorescence. Bars represent 75 μm. Data were presented as mean ± SD (n = 3). Statistical significance: *p < 0.05, **p < 0.01, n.s no significance

Fig. 3

23-HBA inhibited M2 macrophage polarization in a STAT6-dependent manner. A Schematic diagram of experiment. M0 macrophages were treated with siRNA-NC or siRNA-STAT6 for 48 h, and then treated with IL-4 (20 ng/mL) with or without 23-HBA (20 μM) for 48 h. These treated cells were collected for the following analyses. B Relative protein levels of p-STAT6/STAT6 were determined by western blot. β-tubulin was used as internal control. C The expression of CD206 was analyzed by flow cytometry. Data were presented as mean ± SD (n = 3). Statistical significance: *p < 0.05, **p < 0.01, n.s no significance

Fig. 4

23-HBA attenuated M2 macrophage-mediated 5-FU resistance of SW480 cells. A Schematic diagram of experiment. B Dose–response curves showing the sensitivity of SW480 cells to 5-FU in M0-CM and IL-4-CM. C The CCK8 assay was used to measure the relative number of SW480 cells treated with 5-FU (45 µM) for 48 h in M0-CM, IL-4-CM, and IL-4+(10, 20 µM) 23-HBA-CM. D SW480 cells were treated with or without 5-FU (45 µM) in M0-CM, IL-4-CM, and IL-4+20 µM 23-HBA-CM for 48 h, and the percentage of apoptotic cells was analyzed by flow cytometry. Data were presented as mean ± SD (n = 3). Statistical significance: *p < 0.05, **p < 0.01

Fig. 5

23-HBA reversed M2 macrophage-mediated signal response of SW480 cells. A Cytokine array analysis of the conditioned medium from the macrophages. The table in the bottom summarizes the relative signal intensity of the indicated cytokines. B IL-10 levels in M0-CM, IL-4-CM, and IL-4+(10, 20 µM) 23-HBA-CM were determined by ELISA. C SW480 cells were treated with or without 5-FU (45 µM) in M0-CM, IL-4-CM, and IL-4+20 µM 23-HBA-CM for 48 h, and the relative protein levels of p-STAT3/STAT3 and the expression of Bcl-2 protein were analyzed by western Blot. β-tubulin was used as internal control. Data were presented as mean ± SD (n = 3). Statistical significance: *p < 0.05, **p < 0.01

Fig. 6

23-HBA downregulated M2 macrophage-induced 5-FU resistance of SW480 cells in a STAT6-dependent manner. A Schematic diagram of experiment. After transfecting macrophages with siRNA-NC and siRNA-STAT6, SW480 cells were treated with 5-FU (45 µM) for 48 h in M0-CM, IL-4-CM, and IL-4+20 µM 23-HBA-CM. B CCK8 assay was used to measure the relative number of SW480 cells. C IL-10 levels in conditioned medium were determined by ELISA. D The relative protein levels of p-STAT3/STAT3 and the expression of Bcl-2 protein were analyzed by western blot. β-tubulin was used as internal control. Data were presented as mean ± SD (n = 3). Statistical significance: *p < 0.05, **p < 0.01, n.s no significance

Fig. 7

23-HBA attenuated tumor chemoresistance in vivo. A The picture of isolated tumors and the weight of tumors. B Body weight of mice in each group. C Spleen index. D Thymus index. Statistical significance: *p < 0.05, **p < 0.01, n.s no significance

Fig. 8.

23-HBA inhibited M2 macrophage polarization via STAT6 in vivo. A p-STAT6 and CD206 staining of mice tumor tissues, Original magnification was 200×, bars represent 50 μm. B Quantification of immunohistochemistry staining of p-STAT6 and CD206. Data were presented as mean ± SD (n = 3). Statistical significance: *p < 0.05, **p < 0.01, n.s no significance